1. Field of the Invention
The methods and apparatus described are related to analytical spectroscopy, in particular Raman spectroscopy. The methods and apparatus are further related to the discrimination of two closely spaced spectral lines without the use of a spectrometer. The methods are further related to the discrimination and detection of isotopes and isotope ratios using Raman spectroscopy, particularly the detection of metabolic conversion of an isotopically labeled substrate to an isotopically labeled product. In certain embodiments, a Raman shifted frequency of various gaseous compounds (such as carbon 13 labeled carbon dioxide (13CO2)) is distinguished from the corresponding Raman shifted frequency of the naturally abundant isotope using an atomic vapor filter. In other embodiments, a Raman shifted frequency can be employed for nitrogen 15 in NOx or 15NH3. In one embodiment, a Raman shifted frequency can be employed for sulfur in XSO4. In one embodiment, a Raman shifted frequency can be employed for deuterium in HDO or D2O.
2. Background
Raman scattering is a type of inelastic scattering of electromagnetic radiation, such as visible light, discovered in 1928 by Chandrasekara Raman. If a beam of monochromatic light is passed through a substance some of the radiation will be scattered. Although most of the scattered radiation will be the same as the incident frequency (Rayleigh scattering), some will have frequencies above (anti-Stokes radiation) and below (Stokes radiation) that of the incident beam. This effect is known as Raman scattering and is due to inelastic collisions between photons and molecules leading to changes in the vibrational and rotational energy levels of the molecules. This effect is used in Raman spectroscopy for investigating the vibrational and rotational energy levels of molecules. Raman spectroscopy is the spectrophotometric detection of the inelastically scattered light.
xe2x80x9cStokesxe2x80x9d emissions have lower energies (lower frequencies or a decrease in wave number (cmxe2x88x921)) than the incident laser photons. They occur when a molecule absorbs incident laser energy and relaxes into an excited rotational and/or vibrational state. Each molecular species will generate a set of characteristic Stokes lines that are displaced from the excitation frequency (Raman shifted) whose intensities are linearly proportional to the density of the species in the sample.
xe2x80x9cAnti-Stokesxe2x80x9d emissions have higher frequencies than the incident laser photons. Anti-Stokes emissions occur only when the photon encounters a molecule that, for instance, is initially in a vibrationally excited state due to elevated sample temperature. When the final molecular state has lower energy than the initial state, the scattered photon has the energy of the incident photon plus the difference in energy between the molecule""s original and final states. Like Stokes emissions, anti-Stokes emissions provide a quantitative fingerprint for the molecule involved in the scattering process. This part of the spectrum is seldom used for analytical purposes since the spectral features are weaker. However, the ratio of the Stokes to the anti-Stokes scattering can be used to determine the sample temperature if it is in thermal equilibrium.
The Stokes and anti-Stokes emissions are collectively referred to as spontaneous xe2x80x9cRamanxe2x80x9d emissions. Since the excitation frequency and the frequency of the Stokes (and anti-Stokes) scattered light are typically far off the resonance of any component in the sample, fluorescence at frequencies of interest is minimal. The sample is optically thin and will not alter the intensities of the Stokes emissions (no primary or secondary extinctions), in stark contrast to infrared spectroscopy.
Spectroscopy may be used in a variety of diagnostic tests, in particular diagnosis of Helicobacter pylori (H. pylori) infection. H. pylori is a spiral shaped bacterium that lives in the stomach and duodenum, a section of intestine just below stomach. It has a unique way of adapting to the harsh environment of the stomach. The inside of the stomach is bathed in about half a gallon of gastric juice every day. Gastric juice is composed of digestive enzymes and concentrated hydrochloric acid, which can readily tear apart the toughest food or microorganism. The stomach is protected from its own gastric juice by a thick layer of mucus that covers the stomach lining. H. pylori take advantage of this protection by living in the mucus lining and counteracting a local acidic environment with the enzyme urease. Urease converts urea into the acid neutralizing compounds bicarbonate and ammonia. The production of these acid-neutralizing chemicals around H. pylori protect it from the acid environment of the stomach.
H. pylori are isolated from the body""s immune response by the mucus lining of the stomach. The immune system will respond to an H. pylori infection by sending cellular mediators of the immune response and other infection fighting agents. However, the mucus lining of the stomach is not readily accessible to cellular mediators of the immune response. As long as the bacteria are present the immune system continues to respond. Cellular mediators of the immune response die and release superoxide radicals and similar compounds on the cells of the stomach lining. H. pylori can feed on the extra nutrients that are sent to reinforce the cellular mediators of the immune response and within a few days gastritis results, and perhaps eventually a peptic ulcer. It may not be H. pylori itself which causes the peptic ulcer, but the inflammation of the stomach lining; i.e. the response to H. pylori. 
Traditionally endoscopy with biopsy is used to check for ulcers, but recently, non-invasive methods have been developed for detecting H. pylori. Certain conditions and diseases, including H. pylori infection, can be detected non-invasively by analyzing the conversion of a labeled substrate into a labeled product. For instance, air exhaled by persons suspected to be infected by H. pylori can be analyzed to detect the presence of the bacteria in the gastrointestinal tract. In one such method, a patient swallows a radiolabeled urea preparation, 14C labeled urea. H. pylori present in the gastrointestinal tract degrade the urea to ammonia and bicarbonate, the bicarbonate being labeled with 14C. The gastrointestinally formed bicarbonate is converted to 14C labeled carbon dioxide and is transported to the lungs through the normal physiology of the body where it is exhaled together with carbon dioxide formed by other body processes. In one detection method the exhaled carbon dioxide (CO2) is trapped and examined with a scintillation counter to detect the presence of 14CO2 by radioactive decay. A similar method uses the ingestion of 13C labeled urea and a combination of gas chromatography/mass spectroscopy to detect 13CO2 in the breath. In another method of detection infrared spectroscopy is used to detect 13CO2 in the breath.
Current techniques available to the physician for the detection of H. pylori by a breath test are scintillation counting of 14CO2 in breath samples and mass spectrometric analysis of 13CO2 in breath samples analyzed by an off-site central laboratory. These techniques require about one week for analysis. The use of 14C typically requires the presence of a physician trained in nuclear medicine and can not be used in pregnant women and children, as well as being banned in countries such as Sweden and France. These methods entail the submission of a sample to an offsite laboratory, and/or the handling of radioactive materials, which is both time consuming and expensive.